Abstract
Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of 19 widely used fluorophores was conducted by searching 26 comprehensive biomedical and chemical literature databases and analyzing the retrieved material. These fluorophores included Alexa Fluor 488 and 514, BODIPY FL, BODIPY R6G, Cy 5.5, Cy 7, cypate, fluorescein, indocyanine green, Oregon green, 8-phenyl BODIPY, rhodamine 110, rhodamine 6G, rhodamine X, rhodol, TAMRA, Texas red, and Tokyo green. Information regarding cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity was included. Considerable toxicity-related information was available for the Food and Drug Administration (FDA)-approved compounds indocyanine green and fluorescein, but published information on many of the non-FDA-approved fluorophores was limited. The information located was encouraging because the amounts of fluorophore used in molecular imaging probes are typically much lower than the toxic doses described in the literature. Ultimately, the most effective and appropriate probes for use in patients will be determined by their fluorescent characteristics and the safety of the conjugates.
OPTICAL IMAGING has great potential as a clinical tool for cancer diagnosis. The most common optical molecular imaging probes are composed of fluorescent dyes conjugated to targeting ligands, which have affinity for tumor cells. When excited by a particular wavelength, fluorophores emit light at a slightly longer wavelength (ie, the Stokes shift), thus differentiating the targeted, cancerous cells from adjacent normal tissue. Fluorophore conjugates can assist in detecting tumors submillimeter in size in real time, using portable and relatively inexpensive equipment. However, fluorophores are also potentially toxic. To bring a new agent to clinical trials requires the preclinical assessment of an agent's toxicity in multiple species, to guide the determination of a dose at which there are no observed adverse effects. These data are relied heavily upon by the Food and Drug Administration (FDA) when human toxicity is first investigated in a phase I trial. We undertook an exhaustive search of the biomedical, chemical, and toxicology literature for information on 19 fluorophores currently used in molecular imaging probes. The probes chosen were fluorophores we had firsthand experience with or judged to be popular in the molecular imaging community. Focusing on doses resulting in adverse effects and the mechanisms of toxicity, we sought to determine the extent of available published toxicologic data.
Methods
Literature searches were performed for information on 19 organic fluorophores (Figure 1) (Table 1) using multiple online database retrieval systems (PubMed, EMBASE, TOXNET, Scopus, Biological Abstracts, Web of Science) without restrictions on year published, with coverage extending back to 1900 (Table 2). These six primary databases were searched by the two first authors. Additional searching was performed in up to 20 other databases (ChemIDplus, Merck Index records, TOXCENTER, HCAPLUS, CABA, BIOSIS, LIFESCI, PASCAL, RTECS, SciSEARCH, Aquire, BIOTECHABS, CSNB, DISSABS, EMBAL, ESBIOBASE, HEALSAFE, HSDB, MSDS-OHS, and ULIDAT) by the third author because many of these additional, more specific databases had restricted access and required knowledge of a specialized query language (Table 3).
Chemical structures: (A) Alexa Fluor 488; (B) Alexa Fluor 514; (C) BODIPY FL; (D) BODIPY R6G; (E) carboxyrhodamine 6G; (F) Cy5.5; (G) Cy7; (H) cypate; (I) fluorescein; (J) indocyanine green; (K) Oregon green 488; (L) 8-phenyl BODIPY; (M) rhodamine green; (N) ROX; (O) rhodol; (P) TAMRA; (Q) Texas red; (R) Tokyo green.
The general search method in the primary databases was to construct queries seeking the name of the fluorophore or any alternate names in the title, abstract or indexing terms, and one of the following terms anywhere in the database record: toxicity or toxic* (wildcard), safety, or biocompatibility. Also, the fluorophores Tokyo green, TAMRA, cypate, Cy 7, Alexa Fluor 488, and Alexa Fluor 514 were searched without accompanying toxicity-related words to maximize retrieval. This was deemed potentially beneficial because little formal toxicity-related information is available for these compounds. When available, Chemical Abstracts Registry Numbers (CASRNs) were also searched, as well as names in databases with appropriate indexing, such as CAPlus. Furthermore, the initial developers and/or company producing each fluorophore were contacted to request confirmatory data as well as any fluorophore toxicity information of note.
The titles and abstracts retrieved by each of the searches and the Material Safety Data Sheets provided by the manufacturer were reviewed for citations to potentially relevant articles. Relevant articles were selected, and the full text was obtained and examined. These publications were analyzed to obtain information on fluorophore dosage, mechanism of toxicity, and effect on an animal, human, or cellular system. This information was also analyzed to determine known toxic effects and doses and what further toxicity information must be known before the fluorescent compounds can be considered for human use.
Results
For all 19 fluorophores, more than 21,288 articles (including duplicate items, which were returned more than once from separate databases) were returned in the searches. Of these, approximately 240 were selected for detailed analysis; a subset of these provided the relevant information that is cited in this literature review. The toxicologic data collected for each of the fluorophores is summarized in the following sections. Evidence of cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity is included. For some fluorophores, additional toxicity information was available, including median lethal dose (LD50) (or clinical symptom thresholds, which is used in more recent articles); mutagenicity (alters genetic structure); carcinogenicity (potentially induces cancer); teratogenicity (toxic to fetuses); and developmental toxicity. Although the determination of the toxicity of an individual fluorophore is the investigational goal of this study, many of the fluorophores have been investigated only when conjugated to another substance. We do report on the safety and toxicity of those conjugates. Nonhuman species toxicity data are included as many of the fluorophores have not undergone human trials. Concentrations are reported in mg/mL or μg/mL for consistency; the results below are reported in order of decreasing amounts of information retrieved.
Indocyanine Green
Indocyanine green (ICG) was approved by the FDA in 1959 as a contrast agent for retinal angiography. Commercial names include Cardio Green, IC-Green, and Fox Green. ICG toxicity in cells, tissues, animals, and humans is well studied. Specific information on the mutagenicity of ICG, however, could not be located.
Although safe at clinical doses, at a concentration of 2.5 mg/mL, ICG has cytotoxic effects in vitro. 1 It accumulates intracellularly and decreases dehydrogenase activity in the mitochondria. 2 4 In liver cells, ICG inhibits mitochondrial oxygen consumption.5,6 In MIO-M1 cultured human retinal glial cells, 2.5 mg/mL ICG induced apoptosis through both reduction of thymidine incorporation in the MIO-M1 cultured human retinal glial cells and induction of the caspase cascade. 7 An ICG solution of 5 mg/mL is toxic to cultured lens epithelial cells. 8 ICG at a concentration of 0.25 mg/mL in cultured human retinal pigment epithelial (RPE) cells, ARPE19, exposed to light of wavelength 400 to 800 nm for 15 minutes, causes the upregulation of the cell-cycle arrest protein p21 as well as cell-cycle arrest genes bax and p53. 9
ICG has photodynamic toxicities on the inner retina when exposed to light.8,10 It increases the rate of deoxyribonucleic acid (DNA) synthesis, and the toxicity increases with the duration of light exposure. 4 A 0.1 mL dose of ICG at a concentration of 2.5 mg/mL in rabbit eyes causes the loss of photoreceptor outer segments after 10 minutes of endoillumination. 11 The toxicity of ICG is enhanced by the presence of sodium. 12 Photodynamic toxicity has also been attributed to the production of a low level of reactive oxygen species 13 and has been investigated for use in laser-induced photodynamic therapy against infectious agents14,15 and cancerous cells. 16
The intravenous LD50 for mice and rats ranges from 50 to 80 mg/kg and 60 to 87 mg/kg, respectively.8,1719 The intravenous LD50 for rabbits is 300 mg/kg. 19 The typical clinical dose of ICG is 3.572 μg/kg, and the maximum intravenous dose for humans is 5 mg/kg; thus, the clinical dose is approximately 4 log lower than the lowest LD50. 8
Because of its long history of use, there is extensive literature on the toxicity of ICG in humans. ICG is used during retinal surgery at the clinical concentration of 1.25 mg/mL in volumes of about 0.2 mL. 20 However, after intravitreous injection, ICG binds to plasma proteins, which accumulate at the vitreoretinal interface. 21 ICG can cause functional damage and RPE atrophy at 10.0 mg/mL and 5.0 mg/mL concentrations when 0.05 mL is injected into the eye.5,22,23 When ICG leaks through a macular hole into the subretinal space, it causes RPE atrophy at that site.24,25 ICG has been shown to induce apoptosis in RPE cells, as well as gene expression alterations and separation of the inner retina from the internal limiting membrane. 26 ICG is also damaging to glial cells. 27 It injures the nerve fibers at the optic nerve head and can disrupt the activity of myelinated dorsal root axons in rats at a concentration of 0.22 mg/mL in the bloodstream. 28 Studies suggest that ICG should not be used during macular surgery because of its toxicity to the retina, and there is significantly worse visual acuity in groups of patients in which ICG was used to visualize the retina.20,29 Furthermore, if accidentally splashed, ICG is difficult to rinse out of the eye, and if left in the subretinal space for more than 6 months, ICG can cause RPE atrophy, photoreceptor apoptosis, glial and ganglion cell death, and visual field loss results.27,30
Common Fluorophores
CASRN = Chemical Abstracts Registry Number; NA = not available.
These numbers may not match the structure shown but may refer to a commonly used conjugate of fluorophore listed.
Major Widely Accessible Databases
TOXNET is composed of records from other databases: ChemIDplus, HSDB, TOXLINE, CCRIS, DART, GENETOX, IRIS, ITER, LactMed, TRI, Has-Map, Household Products, and TOXMAP.
Databases with More Restricted Access
Idiosyncratic adverse reactions owing to ICG injection at the recommended dose include low blood pressure, itchiness, and pain in the vein.18,31 There have been at least two reported deaths owing to ICG injection resulting from anaphylactic reactions and cardiorespiratory arrest, and the estimated death rate is 0.0003%. 18 ICG does not appear to cross the blood-brain barrier and is excreted unmetabolized by the liver.18,32 Intravenous ICG rapidly binds plasma proteins, leading to prolonged clearance times in vivo.
In summary, ICG is a widely used, FDA-approved fluorescent dye that at much higher doses than used clinically causes in vitro and in vivo toxicity.
Fluorescein
Fluorescein was FDA approved in 1976 as a contrast agent for angiography. Commercial names include Funduscein-25 and Fluorescite. Research regarding fluorescein toxicity to tissues and in vivo is available, but information on the cytotoxicity and mutagenicity was lacking in the literature.
In excised embryonic chicken retina, a concentration of 0.2 mg/mL fluorescein inhibits neuron growth. 33 Fluorescein may irritate the nerve roots when injected intrathecally (within the spinal cord) and affects the central nervous system.34,35 It is also known to cause ataxia (gait disturbance) in rats at a concentration of 4,200 mg/kg. 36
The median lethal concentration (LC50) for turbot fish (Scophthalmus maximus) is 997.1 mg/L for 1 to 4 days of exposure. 35 The oral and intravenous LD50 for mice is 4,738 mg/kg and 2,200 mg/kg, respectively. 36 40 The oral and intravenous LD50 for rats is 6,721 mg/kg and 600 to 1,000 mg/kg, respectively.36,37,39,40 The intravenous LD50 for dogs is 1,000 mg/kg. 37 The oral and intravenous LD50 for rabbits is 2,500 mg/kg and 300 mg/kg, respectively. 39 The typical clinical dose ranges from 7.1 to 10 mg/kg, with 30 mg/kg used for the examination of the anterior segment of the eye.37,39,41
Fluorescein may be a developmental toxin. Fluorescein has been shown to cross the placenta in rats and humans, and in one review of fluorescein angiography performed in pregnant patients, a number of low birthweight babies were reported. 42 The study, however, was not controlled for other risk factors.
Adverse reactions to intravenous fluorescein occur in 4.82% of patients. 43 Of patients with reactions, nausea and vomiting are the most common (10–20% and 1.78%, respectively).43,44 Patients also experience dizziness, headache, diarrhea, convulsions with bradycardia, circulatory problems, irritation at the injection site, vasovagal episodes (fainting), pruritus (itchy), urticaria (in 0.5% of patients with reactions), myocardial infarction, breathlessness, extravasation, thrombophlebitis (inflammation of a clot in the superficial venous system), local tissue necrosis, laryngeal and facial edema, bronchospasm, and anaphylaxis and allergic reactions (in 0.5–1% of total patients). 43 48 The primary route of excretion is the kidney. 38 Cardiac arrest and death are very rare; the mortality rate is 0.0005%.18,46
Fluorescein is a widely used, FDA-approved fluorescent dye. Based on the existing data, it can have toxic effects in cell culture and on patients at high doses.
Rhodamine 6G
Rhodamine 6G (Rh6G) is a non-FDA-approved fluorophore that has demonstrated mutagenicity and toxicity in cells, tissues, and organisms. Rh6G is cytotoxic to Friend leukemia cells and doxorubicin-resistant variant cells, ARN15, in culture, killing 100% of the former at a dose of 0.1 μg/mL and the latter at 10 μg/mL. 49 Rh6G is also lethal to Salmonella TA98 and TA100 at 3.0 mg/plate and 0.5 mg/plate, respectively. 50 Rh6G demonstrates toxicity to tissue, causing severe damage to rat retinal ganglion cells at a dose of 2 μL at 5.0 mg/mL concentration. 51
In mice, Rh6G accumulates in mitochondria and disrupts oxidative phosphorylation and adenosine triphosphate (ATP) synthesis by inhibiting the function of adenosine triphosphatase (ATPase) and the processing of the mitochondrial matrix peptides. 52 At an intravenous dose of 0.5 mg/kg/d for 4 days, ATP synthesis in pregnant mice was reduced by almost half. At a concentration of 10 μg dye/mg protein, 86% of ATPase production in the mice was inhibited. 53
Studies suggest that Rh6G is a mutagen at high concentrations. It induces single-strand breaks in the DNA of Chinese hamster ovary cells at concentrations of 500 to 1,000 μg/plate and is mutagenic to Salmonella strains. 54
Studies also suggest that Rh6G may be a carcinogen. In one study, it potentially caused integumentary keratoacanthomas, pheochromocytomas, and malignant pheochromocytomas in rats that were fed 10 or 12 mg/kg of Rh6G for at least 14 days. 55
Rh6G has demonstrated teratogenic effects in mice as well. 56 Of pregnant mice injected intraperitoneally with 15 mg/kg/d of Rh6G, 61% experienced gross malformations of the fetus. This study also suggests developmental toxicity of Rh6G as 17% of the mice fetuses experienced prenatal mortality and the typical fetal body weight was significantly reduced. 52
Thus, Rh6G is a contrast agent that has demonstrated a host of toxic effects to cells, tissues, and organisms at high doses.
Texas Red
Information on the cytotoxicity, tissue toxicity, in vivo toxicity, and mutagenicity of Texas red is limited. Although studies on the toxicity of pure Texas red were not returned in any search, information on Texas red conjugates was available. When Texas red is conjugated to bovine serum albumin (BSA), it alters the protein's physicochemical characteristics. 57 It causes a minimal yet significant change on the relative charge of BSA but does not influence the size or isoelectric point of the protein. 57 Conjugates of Texas red have shown phototoxic effects in rat erythrocytes exposed to epi-illumination. Of the four BSA-fluorophore conjugates tested in these cells (fluorescein isothiocyanate [FITC], tetramethylrhodamine isothiocyanate [TRITC], BODIPY-FL, and Texas red), Texas red is the third most phototoxic, ranking below FITC and BODIPY-FL. 58 In lamprey larvae, normal brain neuron regeneration when labeled with 62.5 mg/mL Texas red dextran amine, shows no neurotoxic effects. 59
BODIPY FL (Boron-Dipyrromethene)
A BODIPY-verapamil conjugate is toxic to multidrug resistant human KB carcinoma cell lines in culture at concentrations greater than 10 μM (approximately 7.3 μg/mL) and is more toxic than verapamil alone. 60 Another derivative of the BODIPY fluorophore, C11-BODIPY581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid), an oxidation-sensitive fluorescent lipid peroxidation probe, is not toxic to rat-1 fibroblasts in culture at concentrations up to 50 μM (approximately 0.03 mg/mL), highest concentration tested. 61
Cy5.5
The only information located regarding the in vivo toxicity of Cy5.5 is given on the Material Safety Data Sheet; it is harmful if swallowed and may cause allergic respiratory and skin reactions. 62 A study of Cy5.5 conjugated with the caspase-cleavable peptide DEVD, Cy 5.5-DEVD26-PEI-DOCA20, found that the compound is not cytotoxic to HeLa cells at concentrations of 20 μg/mL or less. 62
Cypate
Bioconjugates of cypate are not toxic to animals up to 10 μmol/kg, and Cyp-GRD (cypate conjugated with a linear hexapeptide, GRDSPK) is not toxic to the αv β3-integrin (ABI)-positive tumor cell line, A549, in culture up to a concentration of 100 μM (approximately 0.1 mg/mL).63,64 In general, cypate is considered a “biocompatible” dye. 65
Rhodamine 110
Limited toxicity information is available for rhodamine 110, also commonly known as rhodamine green. Rh110 accumulates in the mitochondria in human lymphoblastoid cells but shows no cytotoxic effects at concentrations up to 10 μM (approximately 5.1 μg/mL). 66 However, it has been found cytotoxic at concentrations greater than 100 μM (approximately 51 μg/mL) and accumulates in Friend leukemia cells, causing cell death. 49
Oregon Green
Information on the cytotoxicity, toxicity to tissues, in vivo toxicity, and mutagenicity of Oregon green is limited. In one experiment, 10 μM (approximately 5.1 μg/mL) Oregon green 488 conjugated with paclitaxel was safely injected intravenously into swine, which were observed for up to 96 hours postinjection and suffered no toxicity-related effects. 67
Tokyo Green
The Tokyo green dyes are an array of recently developed fluorescein derivatives. 68 Information on the tissue toxicity, in vivo toxicity, and mutagenicity of Tokyo green is limited. The toxicity profile of Tokyo green is generally safe and similar to that of fluorescein (unpublished data by Yasuteru Urano, PhD, of University of Tokyo, 2007).
Remaining Dyes Searched
For Alexa Fluor 488, Alexa Fluor 514, BODIPY R6G, Cy7, carboxyrhodamine 6G, 8-phenyl BODIPY, rhodamine X, rhodol, and 6-TAMRA (tetramethyl-6-carboxyrhodamine), no toxicity-related information was returned in any search.
Discussion
Molecular imaging is emerging as a powerful diagnostic tool for cancer. Among the modalities capable of molecular imaging, optical imaging holds great promise for clinical translation and has the advantages of low cost, real-time acquisition, and portability. Optical imaging often uses fluorophores with characteristic emission spectra linked to targeting ligands. Using multiple fluorophores can open possibilities for multiplex imaging (imaging multiple targets simultaneously). However, if fluorophores are to be used in patients, their potential toxicities must first be investigated. Recognizing that fluorophore conjugates may have different toxicity profiles compared with unbound fluorophores, we reviewed the published information on the toxicity of 19 commonly used fluorophores to survey the current state of knowledge.
The databases were selected based on their content coverage and availability. PubMed, EMBASE, TOXNET, Scopus, Biological Abstracts, Web of Science, ChemIDplus, Merck Index records, TOXCENTER, HCAPLUS, CABA, BIOSIS, LIFESCI, PASCAL, RTECS, SciSEARCH, Aquire, BIOTECHABS, CSNB, DISSABS, EMBAL, ESBIOBASE, HEALSAFE, HSDB, MSDS-OHS, and ULIDAT were all searched for articles containing fluorophore toxicity data.69,70 The first six databases cite over 60 million articles and draw references from a broad range of relevant subject areas, including toxicology, human medicine, biomedical science, life science, chemistry, hazardous substances, and environmental health. The remaining 20 databases, available from Chemical Abstracts Service/Science Technology Network (CAS/STN), can be searched using a sophisticated command-based query system; many have additional indexing, are more highly targeted, and can return more specific articles from unique journals and resources that may not have been covered by the six main databases. The combination of these databases facilitated a comprehensive examination of published toxicity information. Approximately 20% of articles were returned multiple times, in each of several separate databases, indicating subject overlap between the databases. However, some searches returned no toxicity-related data. This strongly suggests that there are no published results for toxicity studies conducted on these fluorophores.
The doses of fluorophore or fluorophore-conjugate used in animals and patients are considerably lower than the toxic doses described in the studies we reviewed. For example, a typical clinical dose of ICG is approximately 3.6 μg/kg, whereas the LD50 for mice and rats ranges from 50 to 87 mg/kg, a safety factor of 104. Likewise, the typical clinical dose of fluorescein is about 7.1 mg/kg for a 70 kg human, whereas the LD50 for mice and rats ranges from 300 to more than 2,000 mg/kg. These are comparable to other commonly used clinically approved contrast agents such as gadolinium-diethylenetiramine pentaacetic acid (Gd-DTPA) for magnetic magnetic imaging and isopamidol for computed tomography, both of which have dose to LD50 safety factors of approximately 1/100.71,72 Thus, the “therapeutic index” is large enough to allow for the clinical use of ICG and fluorescein despite their toxic effects at higher doses. Molecular imaging probes may also be administered topically rather than systemically, further lowering the potentially toxic dose and increasing the therapeutic index. Determining the safe dose of the majority of these fluorophores, however, will require further clinical and laboratory investigation.
There were difficulties in performing these searches. One major hurdle was the lack of a CASRN for some of the fluorophores in their unconjugated forms, although many fluorophores had multiple CASRNs for multiple forms with distinct conjugation groups. Clear CASRN identification helps ensure that the appropriate compound is being searched and investigated in some of the databases, particularly those databases from STN, such as CAPlus. It is possible that some potentially useful articles could have been missed owing to problematic CASRN identification. Other difficulties encountered were the formatting of the names or search terms as various formats often produced varied results (eg, Cy5.5 vs cy 5.5) and including as many colloquial or trade names for a fluorophore as possible (such as rhodamine 6G, which is also called basic red 1 and basic rhodamine yellow). There was some ambiguity involved with some of these trade names. For example, rhodol refers to a nonfluorescent compound, p-methylaminophenol sulfate, as well as the fluorophore listed here. There is extensive toxiciology literature on p-methylaminophenol sulfate because it is often used in the cosmetics industry, so it was particularly important to verify the actual compound involved. As many relevant CASRNs and other search terms as possible were included for each fluorophore. Furthermore, to ensure the validity of our search results and to avoid publication bias, the inventors and companies marketing the fluorophores searched were contacted, and their contributions were included.
We have described the known published toxicity of candidate fluorophores for molecular imaging probes. What emerges is the patchwork of information that is publicly available. No standardized toxicity panels have been published for even the major fluorophores. However, in molecular imaging compounds, the fluorophores are conjugated to targeting ligands, which bind to tumor cells in vivo. It is recognized that once a fluorophore is chemically changed in any way, such as conjugating it to a targeting ligand, its toxicity is likely to change. Also, the exact chemical or energetic form of the fluorophore could contribute to its toxicity, for example, the light-activated form of ICG. However, we further recognize that after the compound is metabolized in the body, the pure fluorophore may be released and its own inherent toxicity again becomes relevant.
The current information on the toxicity of these fluorophores is not complete. Although sufficient data regarding the toxicity of the FDA-approved agents ICG and fluorescein are available, there is very little published information on many of the unapproved fluorophores. Many of the experiments described in articles we reviewed concerned toxicity studies of other compounds in which the fluorophore was used as part of a biomarker system. Thus, it was impossible to differentiate the toxicity of the experimental agent from that of the fluorophore. Further toxicity studies of the fluorophores for which there are few data are needed. However, the experience with ICG and fluorescein is encouraging because of the large therapeutic index for these agents and the low doses typically required of molecular imaging probes. Ultimately, the most effective and appropriate probes for use in patients will be determined based on their tissue penetration, specificity, efficiency, fluorescent persistence, and safety.
Conclusion
In this literature review, the published information regarding the toxicity of fluorophores used in molecular imaging probes is described. We found that there was substantial inconsistency in the amount of toxicity data among the fluorophores investigated. However, in those fluorophores for which there was adequate information, there is room for optimism that the dose used for clinical probes will be substantially lower than the doses associated with toxicity. Further research into the toxicity of these compounds is needed to bring the diversity of available fluorophores to the clinic.
Footnotes
Acknowledgments
Thanks to Pamela Sieving for her assistance with manuscript review and to Aruna Kodali for her assistance with researching the following six fluorophores: rhodol, carboxyrhodamine green 6G, BODIPY R6G, 8-phenyl BODIPY, Texas red, and rhodamine X.
Financial disclosure of authors: This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
Financial disclosure of reviewers: None reported.